Prevalence and antibiotics resistance status of Salmonella in raw meat consumed in various areas of Lahore, Pakistan

This study reports the prevalence and antibiotics resistance status of Salmonella detected in raw meat from Lahore, Pakistan. Overall, N = 111 meat samples, were collected from local markets. Salmonella was recovered from 57 (51.35%) samples, including 45.83% of poultry, 60% of buffalo, 64.28% of cow, and 60% of goat meat samples. The predominant Salmonella strains were Salmonella enterica serovars; Typhimurium (45.4%), Typhi (27.2%), and Enteritidis (18.1%), identified by VITEK system and 16S rRNA gene sequencing. The isolates exhibited high resistance to Erythromycin (100%), Cefepime (98.24%), Colistin (94.73%), Azithromycin (92.98%), Tetracycline (87.71%), Polymyxin B (84.21%), Ciprofloxacin (84.21%), Trimethoprim-Sulfamethoxazole (80.70%), Nalidixic Acid (80.70%), Kanamycin (78.94%), Chloramphenicol (77.19%), Streptomycin (71.92%) and Ampicillin (64.91%). While the isolates exhibited more susceptibility to Meropenem (75.43%) and Amikacin (73.68%). N = 8 strains were designated as Multidrug Resistant (MDR) and N = 3 as Extensively Drug-Resistant (XDR) Salmonella. The PCR-based detection of resistance genes revealed the presence of blaTEM-1 gene (100%), catA1 gene (64%), and gyrA gene (18%). The whole genome sequencing (WGS) of two selected strains and subsequent downstream analysis confirmed the strains as MDR and XDR Salmonella enterica serovar Typhi. The study showed that raw meat consumed in Lahore carries a significantly high number of drug-resistant Salmonella.

raw meat is the main source of antibiotic-resistant S. enterica infections in humans 3 .Many antibiotic-resistant serotypes of Salmonella are notorious for disturbing the food chain 1 .Another serious concern for public health is the transmission of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Salmonella through the consumption of contaminated food.Several studies have reported the isolation of MDR Salmonella strains that were also resistant to clinically important antibiotics such as Fluoroquinolones, third-generation Cephalosporins, and Carbapenems, which is now considered as an emerging issue around the world 2,6 .
In order to monitor and control antimicrobial resistance (AMR), many developed countries use a regular AMR monitoring and surveillance system, for instance, National Antimicrobial Resistance Monitoring Systems (NARMS) in the United States 8 .On the other hand, due to the inadequacy of surveillance networks, accurate diagnostics, and laboratory capacity, there is limited monitoring and surveillance system in developing countries, such as Pakistan 9,10 .Over the last few years, several studies conducted in Pakistan were focused on checking the prevalence and antibiotic resistance of Salmonella in raw poultry meat 11 , however, limited data is available for raw mutton and beef 2 .Also, a few studies have reported the detection and incidence of antibiotic resistance genes in Salmonella isolated from raw meat in Pakistan.In this context, the present study was designed to evaluate the prevalence, antibiotic resistance pattern, and screening resistance genes in Salmonella serovars recovered from raw (mutton, beef, and poultry) meat samples collected from different markets in Lahore, Pakistan.

Antibiotics resistance pattern of Salmonella isolates
The antibiotic resistance pattern of Salmonella strains to 21 different antibiotics has been summarized in Table 2 and Fig. 2. The strains were designated as "susceptible", "intermediate", and "resistant" by following the instruction  2).In comparison to all the antibiotics, the resistance against Meropenem was less (1.75%).Moreover, Amikacin in the aminoglycosides group was the most effective antibiotic, with 73.68% of Salmonella strains susceptible to it.However, none of the Salmonella strains could be found to be sensitive to all antibiotics used in this study.While following the CDC criteria, Salmonella isolates phenotypically resistant to Ampicillin, Chloramphenicol, and Sulphamethoxazole-Trimethoprim were designated as MDR strains, and Salmonella strains resistant to the above-mentioned antibiotics along with third-generation Cephalosporin and Fluoroquinolones were designated as XDR strains.Based on the antibiotics resistance pattern, 8 MDR and 3 XDR Salmonella strains were recovered from raw meat in this study, which were only susceptible to Meropenem, making it the only effective treatment option for the infections caused by MDR and XDR Salmonella Spp.(Supplementary Table S3).Figure 3 demonstrates an XDR and an MDR Salmonella strain showing resistance to various antibiotics used in this study.

PCR-based detection of antibiotics resistance genes
The resistant Salmonella isolates were screened for three antibiotic resistance genes (bla TEM-1 , catA1, gyrA) through PCR.The results in Fig. 4 and Supplementary Table S3 indicate that the gyrA gene was only detected in two resistant serovars, i.e. S. typhi and S. typhimurium.While catA1 gene was found in seven resistant strains belonging to three groups of serovars, such as S. typhi, S. typhimurium, and S. enteritidis.On the other hand, each MDR and XDR Salmonella strain harbored the bla TEM-1 gene.Figure 4 summarizes the prevalence of antibioticresistance genes in different Salmonella serovars.The results of amplified products of bla TEM-1 , catA1, and gyrA genes after gel electrophoresis are represented in Fig. 5.

Whole genome sequence (WGS) analysis
The assembled genome of Salmonella enterica strain MDR1-PH8 is composed of 71 Contigs (N50 = 217,896) sizing around 4,836,989 bp with GC content of 52% (Fig. 6).The assembled genome of Salmonella enterica strain XDR1-PG2 is composed of 42 Contigs (N50 = 244,952) sizing around 4,772,161 bp with GC content of 52% (Fig. 7).The genome annotations performed by PATRIC (BV-BRC) are summarized in the Table 3.The strain MDR1-PH8 exhibited ArthoANIu value % = 99.96,while the strain XDR1-PG2 exhibited ArthoANIu value% = 99.98 with the reference Salmonella enterica serovar Typhi (NCBI Taxonomy ID: 90370).The whole genome sequencing (WGS) and subsequent sequence analysis of the strains including genome annotations and average nucleotide identity (ANI) calculation with the reference Salmonella eneterica strain confirmed the identity of both of these strains as Salmonella enterica serovar Typhi.The genome sequence data of both the strains is submitted to NCBI, SRA, under the BioProject: PRJNA985576, GenBank, accession numbers JAUDSV000000000 and JAUDPT000000000, respectively.

Discussion
Salmonella is one of the leading food-borne pathogens that cause food-borne illnesses worldwide.Animalderived food such as meat can serve as a mode of transmission of Salmonella to humans 4 .Lahore is the 2nd largest and a highly populated city in Pakistan.Due to this high population, the demand for food, especially meat and meat products, is also very high.Such circumstances could result in poor food processing and management 12 .This study aimed to determine the prevalence of Salmonella spp. in different raw meat samples collected at different local markets in Lahore.The prevalence of Salmonella in poultry meat was found to be 45.83%, which is similar to the percentage obtained in other studies 10,13 .In poultry meat, higher prevalence of Salmonella were found in poultry gizzard (60%), followed by poultry liver (55.5%), heart (45%), wing (40%), and muscles (36.84%).These results contradicted the research findings of Sodagari et al. 14 , in which prevalence was recorded as 8.3% in poultry gizzard, 21.6% in poultry liver, and 14.1% in poultry heart.In this study, the prevalence of Salmonella in buffalo, cow, and goat meat was 60%, 64.28%, and 60%, respectively, which is higher as compared to other studies 2,3,15 .In general, it was observed, S. typhimurium, S. typhi, and S. enteritidis were the dominant serovars isolated from raw meat.However, comparing the prevalence of Salmonella serovars from different studies could be challenging due to the difference in geographical locations, sampling seasons, nature of samples, isolation methods, and abattoirs and retail shops environment 16 .In comparison with other serovars, S. typhimurium had a high prevalence (45.5%) in our findings which is supported by the study of Khan et al. 17 .S. typhi is a humanspecific serovar, and its detection in food shows hygiene failure and improper handling.Very few studies have reported the prevalence of S. typhi in raw meat 18,19 .However, S. typhi prevalence was 27.2% in highly resistant strains isolated from raw meat in this study.Another dominant serovar was S. enteritidis (18.1%); however,   other studies have reported a high prevalence than the findings in this study 2,20 .In this study, VITEK system was applied to avoid the misidentification of recovered strains as Salmonella, as some other bacteria such as Proteus and Citrobacter have similar growth characteristics and may cause false identification 21,22 .Many studies have described that VITEK system is a reliable method for the correct identification of Gram-negative rods 23 .The WGS results and phylogenetic tree analysis confirmed the identity of the strains as Salomonella enterica serovar Typhi.These results verified the original findings regarding the identity of the strains as Salmonella by VITEK characterization and biochemical analysis.
The study also provides quantitative data on the contamination of raw meat with Salmonella in Lahore city.The most probable number (MPN) of Salmonella per liter/g of meat was obtained mostly in the range of > 11 or 1.2-11, demonstrating a high load of Salmonella in raw meat, unlike the study of Yang et al. 24 , in which MPN/g was found in the range of 0.3-10.Another study conducted in Pakistan reported 3.6 MPN/g of Salmonella, which also shows low numbers than the current study 17 .Different raw meat samples heavily contaminated with Salmonella indicate poor hygiene situations and practices by butchers in different meat processing stages, which is also indicated in other studies 25,26 .Moreover, retail meat shops also lack refrigerators for meat storage which can promote the growth of Salmonella in meat.Another possibility of the presence of Salmonella in cattle could be the ingestion of contaminated food by animals or grazing those plants which might have been irrigated with unprocessed effluents containing fecal matter as fertilizer 27 .The risk of Salmonella-induced foodborne diseases is higher in developing countries as compared to developed countries 6 .
The Salmonella serovars recovered from raw meat in the present study were highly resistant to antibiotics, such as Ampicillin (64.91%),Erythromycin (100%), Tetracycline (87.71%),Colistin (94.73%) and Ciprofloxacin (84.21%), which are widely used as growth promoters and therapeutics in the livestock and poultry industry in Pakistan.This overuse and misuse of antibiotics have caused the transmission of drug-resistant Salmonella to humans via the food chain, which has a potentially negative impact on human health 28,29 .In our study, 21 different antibiotics were tested against Salmonella isolates, and high resistance was recorded against several clinically important antibiotics, such as Cefepime (98.24%),Azithromycin (92.98%),Ciprofloxacin (84.21%),Nalidixic Acid (80.70%),Sulphamethoxazole-Trimethoprim (80.70%),Chloramphenicol (77.19%),Ampicillin (64.91%) and Imipenem (49.12%) (Table 2, Fig. 2).The resistance to Ampicillin, Chloramphenicol, Sulphamethoxazole, Ceftriaxone and Ciprofloxacin in Salmonella is threatening as Carbapenems remain the last drug of choice to treat invasive Salmonella infections 3,30 .However, high resistance to Imipenem (49.12%) was found in this study.The previous studies conducted on raw meat had not reported such a high-level resistance to Azithromycin (92.98%) as detected in this study.The possible reason is the widespread use of Azithromycin for COVID-19 treatment in Pakistan 31 .Interestingly, resistance to antibiotics, which are not commonly used in animal and poultry farming demonstrates the significance of other resistance acquiring mechanisms and sources, for instance, horizontal gene transfer, cross-resistance in the same antibiotic group, animal feed contaminated with resistant environmental bacteria, and antibiotics used to treat human infections 3 .Eleven Salmonella strains isolated in this study were reported as MDR and XDR Salmonella.This is in agreement with several recent studies, which have reported the presence of MDR and XDR Salmonella in raw meat worldwide, as well as in Pakistan 11,32,33 .
Different serovars of Salmonella can possess a number of different antibiotic-resistant genes.However, data about the detection of resistance genes in Salmonella isolated from raw meat is rare in Pakistan.In this study, three antibiotic resistance genes were detected in Salmonella strains recovered from raw meat.Given the PCR results, the bla TEM-1 gene was detected in all isolated resistant strains (100%), which is supported by Li et al. 16 in their study by detecting a 100% prevalence of bla TEM-1 gene.The prevalence of the catA1 gene in all resistant Salmonella strains was 63.6% in our study, predominantly detected in S. typhimurium (Fig. 4).As compared to our study, the prevalence is low (47.5%) in the findings of Li et al. 34 .On the other hand, the gyrA gene was detected in only XDR Salmonella strains with a prevalence of 18.1% (Table S3, Fig. 4), in accordance with a previous study that detected the gyrA gene in 20% of Salmonella isolates 35 .The whole genome sequence (WGS) www.nature.com/scientificreports/analysis, specifically the ANI values of the representative MDR-PH8 and XDR-PG2 strains up to 99.96% and 99.98% respectively with the reference Salmonella enterica serovar Typhi further confirmed the identity of these strains as Salmonella enterica serovar Typhi.The genome size of both the sequenced strains and GC content up to 52% is typically in the range for Gram negative bacteria and the members of enterobacteriaceae.Similarly the genome annotations, the number of CDS and subsystems details generally resemble to that of the Salmonella strains, which helped to confirm the identity of the isolated strains in this study as Salmonella enterica serovar Typhi.Overall this study provides comprehensive and useful data regarding the antibiotic resistance status of Salmonella isolated from raw meat in Lahore.

Sample collection
A total of 111 raw meat samples of various categories including poultry meat, mutton, and beef were collected between September 2021 and August 2022.The samples were randomly collected from meat shops located in eleven different local markets in Lahore (including Faisal Town market, Johar Town market, Wafaqi Market, Main Defense market, Township market, Model Town market, Barkat Market, Gulberg market, Allama Iqbal Town market, Samanabad Market, and Shahdara market), and the sampling was carried out once a week.The raw meat samples from different sources included poultry muscles (19), poultry heart (20), poultry liver (18), poultry gizzard (5), poultry wings (10), buffalo meat (10), cow meat (14), and goat meat (15) [Total n = 111].Sterile plastic bags were used to collect the samples aseptically, which were then labelled and transported in an icebox to the research labs at the Institute of Microbiology and Molecular Genetics within 4-6 h for further processing.

Sample processing for the isolation of Salmonella
Each of the sample was processed in three steps, i.e. pre-enrichment, selective enrichment, and isolation on selective media.For pre-enrichment, 10 g of each raw meat sample was homogenized and aseptically transferred into a flask containing 90 ml sterile Buffered Peptone Water (BPW, Oxoid CM1049) as a pre-enrichment media and incubated at 37 °C for 24 h.After overnight incubation, aliquots from pre-enrichment were transferred into a selective enrichment liquid medium at a ratio of 1:10, i.e. 1 ml of the cultured BPW was inoculated into each test tube containing 10 ml of Selenite-Cysteine Broth (Oxoid CM0699).The tubes were incubated at 37 °C for 24 h.The next day, a loopful from Selenite-Cystine Broth was sub-cultured onto Salmonella-Shigella (SS) agar (Liofilchem 610042), XLT-4 agar (Oxoid CM1061), and MacConkey agar (Oxoid CM0007) by using the streak plate method.These plates were incubated overnight at 37 °C.After incubation, the colonies with desired characteristics were selected and re-cultured to purify the bacteria from mixed cultures.The purified isolates were also cultured on blood agar (Oxoid CM0055).

Identification of Salmonella
The colorless colonies with black centers on SS agar, black or red colonies with a black center on XLT-4 agar, colorless colonies on MacConkey agar, and white/grey non-swarming growth on blood agar were supposed to be Salmonella (as shown in Fig.

Strains identification by VITEK 2 compact system
The polystyrene tubes containing 3 ml saline solution were prepared, in which pure bacterial cultures were mixed to make 0.50-0.63McFarland standard.For gram-negative antibiotic susceptibility (GN-AST), 145 µl of GN-ID McFarland solution was taken and mixed in another tube containing 3 ml saline solution.GN-ID and GN-AST cards were placed in the tubes respectively.Then the racks containing tubes and test cards were placed in the filling chamber of VITEK 2 compact system for loading samples.When the test cards had been filled, the racks were transferred to the other chamber for barcode scanning and cutting the filling straws from the cards, and incubating the cards.After logging into VITEK 2 software on the system, the cassette view and samples showing incomplete status were checked.The accession numbers of the samples were entered and the AST of these samples were linked with their IDs.The results were noted after 18 h.

Enumeration of Salmonella using the MPN method
The three-tube three-dilution Most Probable Number (MPN) method was used to enumerate the load of Salmonella in each sample with some modifications in the protocol of the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) 37 .For this purpose, a 25 g meat sample was homogenized, 225 ml of Buffered Peptone Water (BPW) was added, and incubated for 60 min at room temperature.After an hour, 10 ml of sample and BPW mixture was transferred into 3 empty sterile test tubes.Then, 1 ml and 0.1 ml aliquots of prepared homogenate were transferred into further 3, 3 test tubes having 9 ml of sterile BPW to make the first and second tenfold dilutions, respectively.All the inoculated tubes were incubated overnight at 37 °C.After incubation, a loopful from each tube was streaked onto SS agar and incubated at 37 °C for 24 h.The presumptive Salmonella colonies on each plate were subjected to biochemical testing.After confirmation of the number of positive tubes in each set, MPN/ml was calculated by using the USDA-FSIS MPN table 37 .The enumeration of Salmonella in each sample using the MPN technique was similar to the qualitative method used to isolate Salmonella in this study.

Figure 3 .
Figure 3. Antibiotic resistance pattern of an MDR and an XDR Salmonella strain recovered from raw meat.

Figure 4 .
Figure 4. Prevalence of antibiotic-resistance genes in Salmonella isolates recovered from raw meat in Lahore, Pakistan.

Table 1 .
Recovery rate of Salmonella in various meat sources sold at local markets in Lahore, Pakistan.

MPN values of Salmonella from meat samples In
the majority of tested samples, the Most Probable Number (MPN) of Salmonella per milliliter of the sample was > 11, with 4.2 as the lowest number of Salmonella in the sample, which means that more than 11 most probable number or viable numbers of Salmonella were present in every sample.Some samples had Salmonella loads in the range of 1.2-11 MPN/ml, and a few had contaminated values in the range of 0.03-1.2MPN/ml (Supplementary Table

Table 3 .
The genomic annotation of an MDR and XDR Salmonella typhi strains after whole genome sequencing analysis.
S.1).The presumptive colonies of Salmonella were then analyzed by gram staining and biochemical characterization, which included the catalase test, oxidase test, indole test, Triple Sugar Iron (TSI) test, Sulfide Indole Motility (SIM) test, MR/VP tests, urease test, citrate utilization test, and phenylalanine deaminase test by following the protocols of International Organization for Standardization 36 .